FLP/FRT Recombination from Yeast: Application of a Two Gene Cassette Scheme as an Inducible System in Plants

Phytosensors are plants that are genetically engineered for sensing and reporting the presence of a specific contaminant, including agriculturally important biological agents. Phytosensors are constructed by transforming plants to contain specific biotic- or abiotic-inducible promoters fused to a reporter gene. When such transgenic plants encounter the target biotic or abiotic agent, the specific inducible promoter is triggered and subsequently drives the expression of the reporter gene, which produces a signal for detection. However, several systems lack robustness, rapid induction and promoter strength. Here, we tested the FLP/FRT recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic Arabidopsis and tobacco plants using a β-glucuronidase (GUS) reporter. In this model system, a heat-shock inducible promoter was employed to control the expression of the FLP recombinase gene. Upon heat induction and subsequent active FLP-mediated excision event, the GUS gene was placed in close proximity to the 35S promoter resulting in an active GUS reporter expression. Our results demonstrate that the two gene cassette scheme of inducible FLP/FRT recombination system is functional in tobacco and Arabidopsis, providing additional insights into its possible application in phytosensing such as creating strong readout capabilities

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models

Selective Protection of Human Liver Tissue in TNF-Targeting of Cancers of the Liver by Transient Depletion of Adenosine Triphosphate

Background Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials. Methods Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity. Results PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues. Conclusion Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.ISSN:1932-620

Practice network-based care management for patients with type 2 diabetes and multiple comorbidities (GEDIMAplus): study protocol for a randomized controlled trial

Contains fulltext : 136561.pdf (publisher's version ) (Open Access)BACKGROUND: Care management interventions in the German health-care system have been evaluated with promising results, but further research is necessary to explore their full potential in the context of multi-morbidity. Our aim in this trial is to assess the efficacy of a primary care practice network-based care management intervention in improving self-care behaviour among patients with type 2 diabetes mellitus and multiple co-occurring chronic conditions. METHODS/DESIGN: The study is designed as a prospective, 18-month, multicentre, investigator-blinded, two-arm, open-label, individual-level, randomized parallel-group superiority trial. We will enrol 582 patients with type 2 diabetes mellitus and at least two severe chronic conditions and one informal caregiver per patient. Data will be collected at baseline (T0), at the primary endpoint after 9 months (T1) and at follow-up after 18 months (T2). The primary outcome will be the differences between the intervention and control groups in changes of diabetes-related self-care behaviours from baseline to T1 using a German version of the revised Summary of Diabetes Self-Care Activities (SDSCA-G). The secondary outcomes will be the differences between the intervention and control groups in: changes in scores on the SDSCA-G subscales, glycosylated haemoglobin A level, health-related quality of life, self-efficacy, differences in (severe) symptomatic hypoglycaemia, cost-effectiveness and financial family burden. The intervention will be delivered by trained health-care assistants as an add-on to usual care and will consist of three main elements: (1) three home visits, including structured assessment of medical and social needs; (2) 24 structured telephone monitoring contacts; and (3) self-monitoring of blood glucose levels after T1 in 3-month intervals. The control group will receive usual care. The confirmatory primary analysis will be performed following the intention-to-treat (ITT) principle. The efficacy of the intervention will be quantified using two-level linear regression stratified by type of medical treatment adjusted for baseline values on the SDSCA-G. Secondary analyses will be performed according to the ITT principle. In health economic evaluations, we will estimate the incremental cost-effectiveness ratios. DISCUSSION: We hope that the results of this study will provide insights into the efficacy of practice network-based care management among patients with complex health-care needs. TRIAL REGISTRATION: Current Controlled Trials ISRCTN 83908315 (ISRCTN assigned 25 February 2014)

Enhancing the Thermoelectric Properties of Germanium Antimony Tellurides by Substitution with Selenium in Compounds Ge n

Quenched pseudocubic germanium antimony tellurides (GST compounds) exhibit promising thermoelectric properties. These are related to the nanostructures which can be influenced by varying the composition and the thermal treatment. The substitution of Te by Se in bulk samples of GenSb2Ten+3 with high thermoelectric figures of merit (ZT) is possible over a wide compositional range. This results in solid solution series GenSb2(Te1–xSex)n+3 with 0 < x < 0.75 for n ≥ 7. Se substitution reduces the average lateral extension of the defect layers in quenched samples. This is a consequence of the reduced mobility during the quenching process due to the lower cubic to trigonal phase transition temperatures of Se-substituted samples. Most pronounced for n = 7, Se doping increases the transition temperatures between the nanostructured (pseudo)cubic modification of quenched samples and their layered trigonal phase. This increases the temperature ranges in which the materials can be employed without altering their nanostructures and properties. When Se is introduced, the Seebeck coefficient increases and the thermal conductivity decreases. The ZT value of Ge7Sb2Te8Se2, for instance, increases up to 1.2 at 425 °C, which is 6 times higher than that of Ge7Sb2Te10. Similarly, the ZT value of Ge12Sb2(Te1–xSex)15 increases up to a factor of 2 for x = 0.2 at temperatures below 400 °C. The most promising thermoelectric properties (ZT = 1.2 at 425 °C for n = 7 and ZT = 1.1 at 350 °C for n = 12) are observed for x = 0.2 whereas higher Se substitution rates result in a less pronounced effect. The average structures were determined by powder as well as single crystal X-ray diffraction (SCXRD). The real structure of quenched GenSb2(Te1–xSex)n+3 (0 ≤ x ≤ 0.5; n ≥ 7) bulk material was investigated by high-resolution electron microscopy (HRTEM) with respect to the degree of substitution (x) and correlated with the resulting changes of the thermoelectric properties. The decrease of the lateral extension of the defect layers with increasing Se content as found by HRTEM and electron diffraction is confirmed by SCXRD data for Ge∼5Sb2(Te0.13Se0.87)∼8